Post-transcriptional regulation of mRNAs by microRNAs is a widespread and essential component of gene regulation in animals. microRNAs, in association with Argonaute (Ago) proteins, bind to specific sequences in mRNAs to repress translation and/or reduce the level of the target mRNAs. Individual microRNAs may regulate many different genes, while individual mRNAs can be targeted by multiple microRNAs. Numerous microRNAs are expressed in the mammalian central nervous system (CNS), and microRNAs have been implicated in the regulation of a variety of processes in the CNS, including neurogenesis, cell survival and death, cell fate determination, neuronal differentiation, axon outgrowth, and synaptic function. Signal transduction plays essential roles in CNS development and function. Intracellular kinase cascades, activated by extracellular signals, directly modulate most biological processes, including transcription and translation. However, it is not known whether repression of target mRNAs by Ago/microRNA complexes can be modulated directly by signaling pathways. Phosphorylation of the Ago-2 protein by the p38 MAP kinase/MAPKAP kinase 2 (MAPKAP-K2) pathway alters its subcellular localization, and p38 kinase/ MAPKAP-K2 signaling is known to regulate mRNA stability and translation, as well as other biological processes. Phosphorylation of the Ago/microRNA complex and associated proteins by p38/MAPKAP-K2 may promote or prevent assembly of repressive complexes on specific mRNAs, leading to the modulation of microRNA-mediated repression on specific subsets of targets in neurons. We propose to use a recently developed method for biochemical purification and high-throughput sequencing of mRNA fragments associated with the Ago-2 protein to identify microRNA target sites at which Ago-2/microRNA binding is modulated by activation or inhibition of the p38 pathway in primary cerebellar granule neurons. We will further characterize selected target mRNAs that exhibit modulated microRNA binding. Modulation of microRNA binding and repression by signaling pathways would significantly expand the scope of biological processes that could be regulated by microRNAs, and may also explain why some predicted microRNA target sites fail to mediate repression. Both microRNAs and the p38 signaling pathway have been implicated in multiple diseases of the nervous system, and p38 plays critical roles during responses to cellular stress and/or apoptosis, physiological processes that are also linked to microRNA functions. Demonstrating that microRNA function can be rapidly modulated in the CNS by signaling, independent of microRNA expression levels, would impact both our understanding of microRNA biology in the nervous system, and of the role of microRNAs and p38 signaling in neurological disease.